Download 6 Microbiology of yoghurt and “bio” starter cultures

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6
Microbiology of yoghurt and
“bio” starter cultures
6.1
Introduction
The first bacteriological study of yoghurt was made by Grigoroff (1905) who
observed three different micro-organisms present, namely a diplostreptococcus, a
rod/coccal-shaped Lactobacillus and a rod-shaped Lactobacillus. The same observation was also reported by Lüerssen and Kühn (1908). However, the popularity of
yoghurt could be attributed to Metchnikoff (1910), who postulated the theory that
prolongation of life would follow ingestion of a lactic acid bacterium named as Bulgarian bacillus. The presence of this organism in yoghurt was supposed to inhibit
the growth of putrefactive organisms in the intestine.
The Bulgarian bacillus is, in fact, Thermobacterium bulgaricum (Orla-Jensen,
1931), later designated as Lactobacillus bulgaricus (currently known as L. delbrueckii subsp. bulgaricus). However, Rettger and Cheplin (1921) and Rettger et al.
(1935) found that Thermobacterium acidophilin (Lactobacillus acidophillus) is the
lactic acid bacterium that can establish itself in the intestine, and furthermore, that
the main therapeutic value of yoghurt is observed when L. acidophilus is one of the
bacteria present in the starter culture. The classification of the lactic acid bacteria
by Orla-Jensen (1931) is still recognised as the standard method for distinguishing
these organisms, i.e. the sphere shape was Streptococcus and the rod forms were
Thermobacterium, Streptobacterium and Betabacterium. According to Orla-Jensen
(1931), the yoghurt starter organisms were thermophilic lactic acid bacteria capable
of growing at 40–45°C. These organisms were designated as Thermobacterium bulgaricum, Thermobacterium jugurti (Lactobacillus jugurti) and Streptococcus thermophilus. According to the seventh edition of Bergey’s Manual (1957), all the lactic
acid bacteria were grouped into one family, the Lactobacillacceae, which was subdivided into the Streptococceae (ovoid or spherical in shape) and the Lactobacilleae (rod-shaped). But this classification was reorganised in the eighth edition of
Bergey’s Manual (1974) to give two separate families, the Streptococcaceae and the
Lactobacillaceae, whilst in the latest edition of Bergey’s Manual (1986) the same
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organisms are grouped in different sections. For example, the Gram-positive cocci
consist of two families where the genus Streptococcus is grouped in family II, i.e.
Deinococcaceae. However, the genus Lactobacillus is grouped in a separate section
known as regular, non-sporing, Gram-positive rods. The group-N lactic streptococci
(i.e. the mesophilic type) are now known as Lactococcus species, and S. thermophilus
(i.e. a thermophilic organism) has retained its nomenclature.
6.1.1 Historical background and classification
The taxonomic status of S. thermophilus reported by Orla-Jensen (1931) has fluctuated since the 1980s due to the close relationship between this organism and Streptococcus salivarius and, as a consequence, it was denoted as a subspecies (e.g. S.
salivarius subsp. thermophilus). In 1991, a separate species status was reproposed
on the basis of both genetic and phenetic criteria; for further detail see the reviews
by Hardie and Whiley (1992, 1995). Selected characteristics of S. thermophilus are
shown in Table 6.1. Other characteristics may include:
Spherical or ovoid cell morphology, < 1 mm in diameter and forming chains or
occurring in pairs.
• Absence of growth at 15°C, whilst growth at 45°C may give rise to irregular
cells and segments; most strains are able to grow at 50°C or survive heating for
30 min at 60°C.
• Bacteria are Gram-positive, anaerobic homofermentative lactic acid and
produce l(+) lactate, acetaldehyde and diacetyl from lactose in milk.
• Some strains produce exopolysaccharide (EPS), and require B vitamins and
some amino acids for enhanced growth rates.
• Absence of growth in methylene blue (0.1 g 100 ml-1) or at pH 9.6.
• The cell wall peptidoglycon type is Lys-Ala2–3, and 16S rRNA sequence data have
demonstrated close association between S. thermophilus, S. salivarius and Streptococcus vestibularis.
• A group antigen for serological identification has not been demonstrated (see
also Nour et al., 1989; Ehrman et al., 1992).
•
The situation is different when certain Lactobacillus spp. are considered with regard
to classification and nomenclature. The standard method proposed by Orla-Jensen
(1931) (i.e. Thermobacterium, Streptobacterium and Betabacterium) has been
replaced using group I, II or III in the latest edition of Bergey’s Manual (1986);
however, the history of the group and the redefinitions of the lactobacilli have been
reviewed by Bottazzi (1988), Collins et al. (1991), Hammes et al. (1992), Hertel et al.
(1993), Pot et al. (1994) and Hammes and Vogel (1995). Studies of the guanine plus
cytosine (G + C) content of deoxyribonucleic acid (DNA), DNA–DNA hybridisation and enzyme homology have shown that L. jugurti is a biotype of Lactobacillus
helveticus and there is no reassociation between L. bulgaricus and L. jugurti
(Simonds et al., 1971; Nakamura and Anzai, 1971). The DNA homology between L.
jugurti and L. helveticus is about 80–100%, and the former which is considered to
be a maltose-negative variant of L. helveticus is not recognised any more (London,
1976). However, because of the high phenotypic and genomic similarities between
Lactobacillus delbrueckii, leichmanni, lactis and bulgaricus, only L. delbrueckii
has been retained as a separate species, whilst the other organisms are subspecies.
Both L. lactis and L. leichmanni are grouped as L. delbrueckii subsp. lactis and
Selected characteristics of some lactic acid bacteriaa associated with yoghurt
Streptococcus spp.
L. delbrueckii subsp.
Lactobacillus spp.
Characteristic
G + Cc mean (%)
Lactic acid isomer(s)
Growth at 10/45°C
Requirement for
Thiamine
Riboflavine
Pyridoxal
Folic acid
Thymidine
Vit. B12
Carbohydrate utilisation
Aesculin
Amygladin
Cellobiose
Fructose
Galactose
Lactose
Maltose
Mannose
Melezitose
Melibiose
Raffinose
Ribose
Salicin
Sucrose
Trehalose
a
thermophilus
salivarius
delbrueckii
bulgaricus
lactis
acidophilus
helveticus
jugurtib
37– 40
l(+)
-/+
39–42
l(+)
-/+
49–51
d(-)
-/+
49–51
d(-)
-/+
49–51
d(-)
-/+
34–37
dl
-/+
38–40
dl
-/+
39
dl
-/v
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
-
+
+
-
d
+
d
+
+
d
+
+
-
+
+
d
+
d
+
+
+
+
+
+
+
+
+
+
+
+
+
+
d
d
+
+
d
d
+
+
d
d
d
-
+
+
+
+
+
+
+
+
d
+
-
+
d
d
d
-
d
-
+
+
d
None of the organisms produce gas from gluconate and glucose or NH3 from arginine. b L. jugurti is included for comparative purposes. c Mean % of guanine and
cytosine of DNA.
+, Positive reaction by 90% or more strains; -, negative reaction by 90% or more strains; d, positive or weak reaction by 11–89%;, empty spaces indicate no data available.
Data compiled from Hansen (1968), Rogosa and Hansen (1971), Bergey’s Manual (1974, 1986), Ottogalli et al. (1979), Accolas et al. (1980), Tamime (1990), Hammes
and Vogel (1995) and Hardie and Whiley (1995).
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Table 6.1
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L. bulgaricus is currently known as L. delbrueckii subsp. bulgaricus. Table 6.1
illustrates the overall differences between these various lactobacilli. Other characteristics of L. delbrueckii subsp. bulgaricus are:
•
It is represented in Group I or Aa – the obligately homofermentative lactobacilli;
the letter a indicates the affiliation to the L. delbrueckii group.
• The cells are rods and rounded ends, of 0.5–0.8 ¥ 2–9 mm, and occur singly or in
short chains.
• This organism ferments fewer sugars, produces d(+) lactate and acetaldehyde
from lactose in milk, and some strains produce EPS.
• Slight growth occurs at < 10°C and most strains are able to grow at 50–55°C.
• The cell wall peptidoglycan type is Lys-dAsp (see also Park et al., 1991; Sungil
et al., 1996).
In view of the wide range of technical data available on yoghurt and other dairy
starter cultures, it is recommended that the reader consults some selected publications for general information (Accolas and Auclair, 1983; Auclair and Accolas, 1983;
Bianchi-Salvadori, 1983; Sriranganathan et al., 1985; Chassy, 1986; Terre, 1986;
Marshall, 1986, 1987, 1993; Kashket, 1987; Daly, 1987; IDF, 1988a; Roginski, 1988;
Lücke et al., 1990; Schleifer et al., 1991; Gasser, 1994; Roussis, 1994; Stiles, 1996).
According to Mitsuoka (1992), L. acidophilus was first isolated from faeces of
bottle-fed infants and named Bacillus acidophilus, but in 1959, Rogosa and Sharpe
gave a detailed description of L. acidophilus based on their own observations and
those of Tittsler et al. (1947) and Rogosa et al. (1953). Later, Lerche and Reuter
(1962) subdivided the species into five biotypes based on fermentation patterns of
trehalose, melibose and raffinose, while Mitsuoka (1969) expanded the number of
biotypes to ten based on variations in the fermentation of ribose and lactose. More
recently, the phylogenetic approach based on 16S rRNA adopted by Collins et al.
(1991) and Fujisawa et al. (1992) has cast doubt on some of these earlier groupings
but, even so, the identity of L. acidophilus as proposed by Gasser and Mandel (1968)
remains intact. As a consequence, the description of the species by Hansen and
Mocquot (1970) based on a specific strain (ATCC 4356) is still valid.
The taxonomic status of L. acidophilus has not fluctuated over the years and
whilst some characteristics of this organism are shown in Table 6.1, some other
aspects may include:
•
•
•
•
•
•
It is presented in Group I or Aa – the obligately homofermentative lactobacilli;
in the same group as L. delbrueckii subsp. bulgaricus.
The cells are rods with rounded ends, of 0.6–0.9 ¥ 1.5–6 mm, occurring singly, in
pairs and in short chains; cells are non-motile and non-sporulating and proteins
in the cell wall may be important in attaching the bacterium to the intestinal
wall (Bhowmik et al., 1985; Brennan et al., 1986).
This organism requires riboflavin, pantothenic acid, folic acid and niacin for
growth, but not the other B vitamins.
Recent studies (i.e. electrophoresis of cellular proteins or lactate dehydrogenase
and DNA–DNA reassociation) suggest that L. acidophilus strains include six
genomospecies.
No growth occurs at < 15°C, most strains grow about 35–45°C and the optimum
pH for growth is 5.5–6.0.
The cell wall peptidoglycan type is Lys-dAsp.
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According to Mital and Garg (1992), the growth requirements of most strains of L.
acidophilus are quite complex and, as the normal habitat of L. acidophilus is
attached to the walls of the small intestine of mammals, such requirements can
usually be met quite easily. The ability of the species to utilise carbohydrates in vitro
is shown in Table 6.1 and although L. acidophilus is the best known of the healthpromoting lactobacilli, other species of human intestinal origin are often used in fermented milk and comparable data for some of these species has been included as
well. In addition, strains of L. acidophilus may require fatty acids, minerals, peptides
and amino acids, nucleic acid derivatives and vitamins of the B-complex to grow
successfully and, given these requirements, it is not surprising that most strains grow
only poorly in bovine milk. The final value of lactic acid is within the range of 0.3–1.9
g 100 g-1 lactic acid suggested by Rasic and Kurmann (1978) but, while some strains
can secrete these high levels of acid, few strains are sufficiently acid tolerant to
survive such conditions for more than a few days; the optical rotation of the lactic
acid is dl.
The alleged health-promoting properties of L. acidophilus are discussed elsewhere and it is relevant that, in addition to secreting lactic acid, some strains of the
species may produce antibiotic-like substances as well. Some authors have suggested
that such compounds could be important in preventing the growth of pathogens in
the intestine (Shahani et al., 1976), but it could be that intrageneric activity could
be equally relevant. Thus, Barefoot and Klaenhammer (1983) and Barefoot et al.
(1994a, b) purified a bacteriocin compound from a strain of L. acidophilus and found
it to be active against a range of other Lactobacillus spp. If this inhibitory activity
happens in the intestine as well, then it might provide an additional mechanism
whereby indigenous strains of L. acidophilus could retain dominance on the epithelial surfaces.
However, the taxonomic and nomenclature situation of the bifidobacteria have
changed, and in the eighth edition of Bergey’s Manual (1974) they were classified
as Lactobacillus spp., whilst in latest edition of Bergey’s Manual (1986) the same
organisms are grouped in a different section, and known as Bifidobacterium spp.
Currently, 30 different strains of bifidobacteria have been identified which have
been isolated from different sources such as the faeces of humans, animals, birds
and sewage, the human vagina, bees and dental caries. Only six species of bifidobacteria have attracted attention in the dairy industry for the manufacture “bio”
ferment dairy products. These organisms are known as Bifidobacterium adolescentis, breve, bifidum, infantis, lactis and longum, and these species have been isolated
from human subjects for the manufacture of fermented milk. This restriction is
based on the assumption that, if an isolate is of human origin, then it should become
implanted on the walls of, and/or metabolise in, the colon of another human. The
validity of this idea remains open to debate, for there is some evidence that, while
an ingested strain may dominate the colon walls of a patient with a low count, the
strains that are indigenous to that patient will, in time, overgrow the invading
culture. It is relevant also that non-human strains of Bifidobacterium animalis can
adhere to human cells in tissue culture, so that the question of which species should
be permitted in bio-yoghurts is a matter of some debate.
The differentiating characteristics have been reviewed in Bergey’s Manual (1986)
and by Biavati et al. (1992), Sgorbati et al. (1995), Tamime et al. (1995), Kok et al.
(1996), Meile et al. (1997) and Ballongue (1998). Other characteristics may be
considered.
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•
•
•
•
•
•
Yoghurt Science and Technology
Bacteria are Gram-positive, anaerobic heterofermentative, non-motile, nonspore-forming rods (0.5–1.3 ¥ 1.5–8 mm).
Cell morphology of these bifidobacteria grown anaerobically in trypticasephytone-yeast (TPY) medium have distinctive shapes and arrangements (e.g.
“amphora-like”; specific epithet, thin and short; very elongated, thin with slight
irregular contours and rare branching).
The cell wall peptidoglycan varies among the species, but this complex material
consists of linear chains of N-acetylmuramic acid and N-acetylglucosamine molecules alternating along the length of the chain.
Different species utilise different types of carbohydrates (see Table 6.2) and such
fermentations are used for identification purposes. One key enzyme involved is
fructose-6-phosphate phosphoketolase (F6PPK) known as “bifidus shunt”, and
this enzyme can be used to identify the genus; it should be noted that not all
strains produce enough F6PPK for it to be detectable. The fermentation of two
molecules of glucose leads to two molecules of lactate and three molecules of
acetate.
The guanine plus cytosine molecular percentage of the DNA of this genus ranges
between 54 and 67.
A wide range of components have been identified as bifidogenic growth
stimulators.
The rods of bifidobacteria often have an irregular shape, with a slightly concave
central region and swollen ends (i.e. having the appearance of a dog’s bone in a
Disney cartoon). It is, however, not unusual to encounter cells that are coccoid or
appear as very long or short bacilli of varying widths, or the cells may be V, Y or
X-shaped depending on the constituents of the medium on which the colony is
growing. It is believed that, under adverse growing conditions, the cell morphology
changes to produce more branched cells; for example, in a medium deficient in
b-methyl-d-glucosamine, the cells become more branched, while the addition of
certain amino acids (e.g. serine, alanine or aspartic acid) can transform X- or Yshaped cells into curved rods (Glick et al., 1960). Similarly, Samona and Robinson
(1994) transformed coccoid cells of B. bifidum into the Y-shaped form through
the addition of sodium chloride to a medium, but noted that neither B. longum
nor B. adolescentis reacted in the same way. The same authors recorded also
that the pattern of carbohydrate fermentation changed as the morphology altered,
suggesting perhaps that the permeability of the cell membrane to certain sugars
was being modified in parallel with the structural changes taking place in the
wall.
Notwithstanding this tendency of some species to alter in shape, the cell
morphology of species of bifidobacteria grown anaerobically in stabs of TPY extract
medium showed a tendency to adopt distinctive cellular shapes. For example, B.
bifidum forms groups of amphora-like cells, the cells of B. breve are the thinnest and
shortest among bifidobacteria, while B. longum appears as very elongated, relatively
thin cells with slightly irregular contours.
A summary of cell wall and DNA contents of the important species of bifidobacteria species are shown in Table 6.2. The principal component of the cell wall is peptidoglycan, also known as murein. This is a macromolecule that consists of linear
polysaccharide chains (glucose, galactose and rhamnose) which are linked to each
other by tetrapeptide bridges (Ballongue, 1998).
Some selected characteristics of bifobacteria used for the manufacture of bio-yoghurta
Bifidobacterium spp.
Characteristic
G + C mean (%)
Type of
peptidoglycan
Carbohydrate utilisation
Arabinose
Cellobiose
Fructose
Galactose
Gluconate
Inulin
Lactose
Maltose
Mannitol
Mannose
Melezitose
Melibiose
Rafinose
Ribose
Salicin
Sorbitol
Starch
Sucrose
Trehalose
Xylose
a
adolescentis
bifidum
breve
infantis
lactisb
longum
58.9
Lys(Orn)-dASP
60.8
Orn(Lys)-dSer-d-ASP
58.4
Lys-Gly
60.5
Orn(Lys)-SerAla-Thr-Ala
61.9
Lys(Orn)-Ala(Ser)-Ala2
60.8
Orn(Lys)-SerAla-Thr-Ala
+
+
+
+
+
d
+
+
d
d
+
+
+
+
d
+
+
d
+
+
+
+
d
d
-
d
+
+
d
+
+
d
+
d
+
+
+
d
+
d
-
+
+
d
+
+
d
+
+
+
+
d
d
+
+
+
+
+
+
+
+
+
+
+
d
+
+
+
+
+
d
+
+
+
+
+
d
For identification of symbols see Table 6.1. b After Kok et al. (1996) and Meile et al. (1997).
Data compiled from Bergey’s Manual (1986), Biavati et al. (1992) and Sgorbati et al. (1995).
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Table 6.2
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6.1.2 Modification of starter cultures
The characteristics of the various species shown in Tables 6.1 and 6.2 are based
essentially on what are referred to as type cultures. These are strains of the species
that have been: (a) isolated and grown as pure cultures in one of the internationally recognised culture laboratories, (b) examined for a range of characteristics, such
as temperature of growth and/or rate of acid production (Zanatta and Basso, 1992),
fermentation of selected sugars (Hickey et al., 1986), enzyme profiles (BianchiSalvadori et al., 1995), DNA base-pair characteristics (Sriranganathan et al., 1985),
DNA hybridisation reactions (Lick et al., 1996), plasmid homology and/or profiles
(Girard et al., 1987; El-Soda et al., 1989) and DNA fingerprinting (Ramos and
Harlander, 1990), and then (c) designated as a distinct species.This procedure means
that there is held somewhere in a deep frozen (-196°C) or freeze-dried state, a
culture which displays all the characteristics of one recognised species and, once
these characteristics have been recorded in an authoritative reference source (e.g.
Bergey’s Manual, 1986) anyone in the dairy industry or elsewhere can identify, with
a reasonable degree of certainty, any cultures that may be isolated from cheese or
a fermented milk.
For many years, this approach to bacterial taxonomy has worked well, but since
about 1990, the degree of strain variability within species has increased because
taxonomists have begun to employ increasingly sophisticated techniques for
identification, for example, 16S RNA sequencing (Davidson et al., 1996) and the use
of DNA probes to isolate individual strains (Delley et al., 1990; Colmin et al., 1991;
Neve and Soeding, 1997), and the number of cultures available from commercial
suppliers has increased.
Some of this variability has arisen as a natural process of change, because the
selective pressures on a culture of S. thermophilus employed in a dairy in the Middle
East, for example, might well be different from those operational in a plant in North
America (Nunez de Kairwuz et al., 1983; Yoast et al., 1994; Teixeira et al., 1994). The
same species isolated from a cheese factory in Italy might well be different again,
so that the precise definition of a species becomes, in some respects, more difficult
(Sandine, 1987; Mercenier and Lemoine, 1989). A good example of this situation can
be found for the mesophilic starters for cheese, in that while the type culture of Lactococcus lactis subsp. cremoris differs widely from Lactococcus lactis subsp. lactis
with respect to the sugar fermentation pattern, a culture of L. lactis subsp. cremoris
purchased today may well display the same sugar utilisation profile as L. lactis subsp.
lactis (de Vos, 1996).
Although this complicated situation may, in part, be the result of culture evolution as a result of mutation (Mollet and Delley, 1990; see also Germond et al., 1995),
conjugation (Kleinschmidt et al., 1993; Soeding et al., 1993), transformation (Mollet
et al., 1993b) and intercellular and/or plasmid transduction (Mercenier et al., 1988a,
b; Heller et al., 1995; Neve and Heller, 1995a, b), the deliberate genetic manipulation of cultures has become increasingly important (Yu et al., 1984; Chassy, 1987;
Romero et al., 1987; Knol et al., 1993a, b; Sasaki, 1994; Mercenier et al., 1994). Thus,
genetic engineering or recombinant DNA technology can now be employed to
modify the properties of various organisms to generate genetically modified organisms (GMOs) (Herman and McKay, 1986; de Vos and Simons, 1988; Somkuti and
Steinberg, 1988, 1991; Lee et al., 1990b; Gasson, 1997).
To avoid potential conflicts with consumers, bacteria to be used in the manufacture of foods should be subject only to so-called food grade genetic modifications,
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397
which means that the GMO must contain only DNA from the same genus and, possibly, small stretches of imported DNA (Johansen et al., 1995). Thus, a Lactococcus
GMO would only contain DNA from the genus Lactococcus plus a small amount
of imported DNA (Mollet et al., 1993a; Griffen and Gasson, 1995). These small
stretches of non-lactococcal DNA are usually no longer than 50 base pairs and act
as recognition sites for the restriction enzymes used in the actual construction
process (Solaiman and Somkuti, 1991, 1995, 1997a–c; Somkuti and Solaiman, 1997;
Satoh et al., 1997). It is essential, of course, that none of the imported DNA should
provide a code for RNA, and specific DNA probes should be constructed to check
that no additional genetic material has been introduced (Lick and Teuber, 1992).
However, pressure is mounting within the dairy industry for permission to
exchange DNA between any genus of micro-organism associated with food fermentation (Langella and Chopin, 1989), provided that the donor bacterium can be
described as generally recognised as safe (GRAS). Whether or not it is appropriate
for microbiologists to borrow this definition from the chemists has not been challenged, but it is relevant that food-grade GMOs can usually be used in the United
States without specific regulatory approval.
6.1.3 Potential genetic modifications
Genes can be deleted from a strain to avoid the release of an undesirable metabolic
product into a food, or the gene can be replaced with the homologous gene from
another strain (Sasaki, 1994; Ito and Sasaki, 1994). For example, if a strain of Lactococcus has a particularly useful characteristic, such as the secretion of a desirable
flavour component, but the level of b-galactosidase activity is low, then this latter
deficiency could be corrected by introducing a more active copy of the gene from
another strain (Yu et al., 1983; Kochhar et al., 1992). Genes could be inserted into a
strain to expand the range of carbohydrates utilised (Branny et al., 1993, 1996) or
increase resistance to a wider spectrum of bacteriophage or, alternatively, a useful
gene within the existing genome can be copied, so doubling the beneficial activity
(Mollet and Delley, 1991).
An example of the potential offered by these techniques relates to the production of diacetyl, a major flavour component of buttermilk and kefir and a compound
that is usually derived via pyruvate. If genes coding for a-acetolactate synthase, an
enzyme involved in the conversion of pyruvate to diacetyl, could be inserted into a
food-grade culture, diacetyl production would increase and the same approach
could be employed in the synthesis of EPS by S. thermophilus or L. delbrueckii
subsp. bulgaricus. The relevant genes have been identified from several strains and
GMOs with altered texture-producing properties could be constructed (Gasson,
1997).
Exactly how far and fast the construction of GMOs will proceed – or will
be allowed to proceed – remains to be seen, but it seems likely that: (a) the
identification of species within starter cultures is going to become increasingly
imprecise as the borderlines between, for example, L. delbrueckii subsp. bulgaricus
and L. delbrueckii subsp. lactis become blurred as a result of genetic manipulation,
and (b) future generations of yoghurt makers will be able to request the supply of
starter cultures with quite specific characteristics.
In view of the wide range of technical data available on the genetic modifications
of the yoghurt and bio starter cultures, it is recommended that the reader consults
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some selected publications for general information (Nicholson and Sanders, 1988;
le Bourgeois et al., 1989; Schmidt et al., 1989; Miteva et al., 1991; Yohda et al., 1991;
Schroeder et al., 1991; Leong-Morgenthaler et al., 1991; Janzen et al., 1992; Pébay et
al., 1992; Delcour et al., 1993; Poolman, 1993; Mustapha et al., 1995).
6.2
Characteristics of growth
Yoghurt and the many fermented milks known across the world have been traditionally made by the spontaneous growth of indigenous micro-organisms present in
milk. At present, carefully controlled microbial processes have been developed
using selected combinations of cultures and the technology required for large-scale
production has evolved from the knowledge of the physiology and biochemistry of
the micro-organisms involved (refer to Chapter 7).
Since the late 1970s much work has been done on the biochemistry and molecular
biology of S. thermophilus and L. delbrueckii subsp. bulgaricus. Catabolism is not
the only important consideration for a successful fermentation to produce yoghurt
of good quality in terms of flavour and stability, but anabolic pathways also have a
role in providing texture-modifying polysaccharides and providing other compounds which have preservative and health-promoting properties.
6.2.1 Milk as a medium for microbial growth
Lactic acid bacteria are widely distributed in nature and their nutritional requirements are very complex. Table 6.1 shows the fermentation ability and growth
temperatures of the yoghurt starter cultures and some of these characteristics
are used to differentiate the genera and species. S. thermophilus and L. delbrueckii
subsp. bulgaricus and many other lactic acid bacteria are unable to synthesise
a full complement of amino acids and this deficiency dictates their natural
habitat. Milk is a nutritionally rich medium which will support the growth of
many micro-organisms, but the processing of milk provides control over the type of
growth necessary to achieve a desirable product (see Chapter 2; Chandrakanth et
al., 1993).
The metabolic activity of an organism is indicative, to some extent, of its growth
rate, and one of the most popular tests for monitoring starter cultures is the development of acidity in the growth medium. Autoclaved, reconstituted skimmed milk
(10–12 g total solids (TS) 100 g-1) is mainly used and the milk must be free from any
inhibitory substances, for instance antibiotics.The activity of a typical yoghurt starter
culture and the isolated strains of S. thermophilus and L. delbrueckii subsp. bulgaricus is illustrated in Fig. 6.1 which shows a marked difference in the rate of acid
development by the mixed starter compared with the isolated single strains. It is
also noticeable that the rate of acid development of S. thermophilus and L. delbrueckii subsp. bulgaricus increases with increase in incubation temperature, up to
maxima of 40°C and 45°C, respectively; the former organism is initially more active
than L. delbrueckii subsp. bulgaricus in relation to acid production. Although the
activity of mixed strains is optimum at 45°C, it is recommended that, in order to
maintain and/or achieve a ratio of 1 : 1 between S. thermophilus and L. delbrueckii
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Rate of acid development % lactic acid
Microbiology of yoghurt and “bio” starter cultures
Lb. delbrueckii subsp. bulgaricus
Str. thermophilus
1.0
399
Mixed culture
▲
▲
▲
0.8
▲
▲
0.6
▲
▲
▲
▲
0.4
▲
▲
▲
▲
▲
0.2
0
2
4
6
8
0
2
▲
▲
4
▲
▲
▲
▲
6
8 0
2
4
6
8
Time of incubation in hours
Fig. 6.1 Behaviour of single and mixed strain yoghurt cultures propagated at different
temperatures in autoclaved skimmed milk (10 g TS 100 g-1) at 2 ml 100 ml-1 inoculation rate
, 30°C; , 35°C; , 40°C; , 45°C; , 50°C.
•
Note: Test organisms is Chr. Hansen’s (CH-1).
Adapted from Tamime (1977a).
subsp. bulgaricus, the organisms should be propagated together at 42°C using a
2 ml 100 ml-1 inoculation rate (Kurmann, 1967; Tamime, 1977a) or direct-to-vat
inoculation (DVI).
6.2.2 Associative growth
The growth association between the two organisms (S. thermophilus and L. delbrueckii subsp. bulgaricus) of the yoghurt starter culture used to be termed a symbiosis and this relationship has been reported by many workers; the earliest record
dates back to the work of Orla-Jensen (1931). This association could be briefly
described as each organism providing compounds which benefit the other. Since
both S. thermophilus and L. delbrueckii subsp. bulgaricus can grow in milk as single
cultures, the term symbiosis should be replaced by associative growth instead. Pette
and Lolkema (1950a) observed that the rate of acid development was greater when
mixed yoghurt cultures of S. thermophilus and L. delbrueckii subsp. bulgaricus were
used compared with the single strains (see Fig. 6.2; Lee et al., 1990a). Furthermore,
they also observed that the numbers of S. thermophilus, as recorded by the Breed
smear method, were much higher in mixed cultures than when the organism was
grown alone, although no such differences in numbers of L. delbrueckii subsp. bulgaricus were noted. This observation was not true with respect to L. delbrueckii
subsp. bulgaricus as reported by Tamime (1977b). The findings of Pette and Lolkema
(1950b) led them to postulate that the interaction between these two organisms was
mainly dependent on the production of valine by L. delbrueckii subsp. bulgaricus.
However, due to variations in the chemical composition of milk during the year,
other amino acids may also be deficient and hence Pette and Lolkema (1950c) suggested that during the spring months, S. thermophilus required amino acids leucine,
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Yoghurt Science and Technology
Rate of acid development % lactic acid
400
Mixed culture
1.0
0.8
▲
▲
Str. thermophilus
▲
0.6
▲
▲
0.4
Lb. delbrueckii subsp. bulgaricus
▲
▲
▲
0.2
0
2
4
6
8
Time of incubation in hours
Fig. 6.2 Behaviour of single and mixed strain yoghurt cultures propagated at 40°C in
autoclaved skimmed milk (10 g TS 100 g-1) at 2 ml 100 ml-1 inoculation rate
Note: Test organism is Chr. Hansen’s (CH-1).
Adapted from Tamime (1977a).
lysine, cystine, aspartic acid, histidine and valine. During the autumn/winter months,
glycine, isoleucine, tyrosine, glutamic acid, methionine, as well as the six amino acid
mentioned above, were essential.
Bautista et al. (1966) also investigated the associative growth theory and supported the view that L. delbrueckii subsp. bulgaricus stimulates S. thermophilus by
releasing glycine and histidine into the growth medium; they concluded that histidine rather than valine was the most important requirement. However, the stimulation by glycine and histidine, as reported by Bautista et al. (1966), was very poor
in comparison with the various amino acids observed by Pette and Lolkema (1950b).
Accolas et al. (1971) reported that the stimulation of S. thermophilus by milk culture
filtrate of L. delbrueckii subsp. bulgaricus was due to the presence of valine, leucine,
isoleucine and histidine. Bracquart et al. (1978) and Bracquart and Lorient (1979)
concluded that depleting the growth medium of valine, histidine, glutamic acid, tryptophan, leucine and isoleucine reduced the stimulation of S. thermophilus by 50%.
Similar findings were reported by Higashio et al. (1977a), where methionine was also
included as a stimulant amino acid; however, by far the most effective amino acid
was valine (see also Shankar, 1977; Shankar and Davies, 1978; Hemme et al., 1981;
Rao et al., 1982; Marshall, 1983).
It is well established that L. delbrueckii subsp. bulgaricus possesses more proteolytic enzymes than S. thermophilus (see Chapter 7; Rajagopal and Sandine, 1990;
Abu-Tarboush, 1996) and El-Soda et al. (1986) reported that crude cell-free extracts
of the yoghurt lactobacilli stimulated the growth of S. thermophilus; they concluded
that acid production was enhanced by the addition of peptone, amino acids and, to
a lesser extent, water-soluble vitamins, purines and pyridines. A similar view was
reported by El-Abbassy and Sitohy (1993) and Neviani et al. (1995), whilst Carmi-
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Microbiology of yoghurt and “bio” starter cultures
401
nati et al. (1994) concluded that a skimmed milk medium deprived of soluble nitrogen inhibited the growth of S. thermophilus. Other amino acids, which are not the
result of proteolysis by the yoghurt organisms, that have stimulated the growth of
S. thermophilus are: (a) peptides containing lysine (Desmaseaud and Hermier,
1972), (b) hepta- or pentapeptides containing histidine and free non-aromatic amino
acids (Desmaseaud and Hermier, 1973; Hayashi et al., 1974), (c) tripeptides containing histidine, methionine and glutanic acid (Bracquart and Lorient, 1979), (d)
casein hydrolysate (Marshall and Mabbitt, 1980; Marshall et al., 1982; Nakamura et
al., 1991), and (e) the addition of magnesium (Amouzou et al., 1985). However, the
transport of branched amino acids in S. thermophilus is energy dependent and
optimum activity was between 30°C and 45°C for leucine, valine and isoleucine
(Akpemado and Bracquart, 1983). Other technical data available on the associative
growth of the yoghurt organisms have been reported by Radke-Mitchell and
Sandine (1984), Matalon and Sandine (1986), Juillard et al. (1987), Berkman et al.
(1990), Kneifel et al. (1993) and Oberg and Broadbent (1993) (see also Champagne
et al., 1990; Klaver et al., 1992; Franzetti et al., 1997).
Thus, the streptococci benefit from the stronger activity of the lactobacilli and in
return provide certain compounds which stimulate the growth of L. delbrueckii
subsp. bulgaricus. However, glutamic acid uptake in S. thermophilus was energy
dependent (e.g. lactose, glucose and sucrose), but aspartic acid exhibited an
inhibitory effect (Benateya et al., 1986; Bracquart et al., 1989).
Galesloot et al. (1968) investigated the opposite side of the associative growth
relationship between S. thermophilus and L. delbrueckii subsp. bulgaricus. They concluded that, under anaerobic conditions, the former organism produces a stimulatory factor for L. delbrueckii subsp. bulgaricus that is equal to or can be replaced
by formic acid. Furthermore, the same workers looked at the effect of various heat
treatments on milk, and found that in intensively heated milk (i.e. autoclaved and
UHT) the stimulation was masked on account of a compound which could be
replaced by formic acid. However, after the normal heat treatment of milk used for
yoghurt manufacture (e.g. 85–90°C), L. delbrueckii subsp. bulgaricus definitely needs
the stimulatory factor produced by S. thermophilus. The normal presence of this
stimulatory factor in autoclaved milk (Auclair and Portman, 1957; Shankar, 1977;
Marshall, 1983), appears to have been overlooked by both Pette and Lolkema
(1950b) and Bautista et al. (1966).
The production of formic acid by S. thermophilus was confirmed by Veringa et al.
(1968), and Bottazzi et al. (1971) demonstrated that the presence of formic acid
in milk increases the ratio of rods to cocci at concentrations between 30 and
50 g ml-1. This compares with the stimulation of L. delbrueckii subsp. bulgaricus by
formate at 20–30 mg ml-1 (Galesloot et al., 1968; Shankar, 1977; Marshall, 1983) and
40–600 mg ml-1 (Accolas et al., 1971; Pulsani and Rao, 1984; Kikuchi et al., 1985; ElAbbassy and Sitohy, 1993; Moreira et al., 1997). This variation in the level of formate
required to promote activity could be attributed to the use of different strains of L.
delbrueckii subsp. bulgaricus. Also, the amount of formate production by S. thermophilus is dependent on strain, culture medium and growth temperature (Perez
et al., 1990, 1991); the streptococci produce formic acid in milk only if the level of
oxygen £ 4 mg O2 l-1 (Driessen et al., 1983).
Some L. delbrueckii subsp. bulgaricus strains grown in milk heated to 100°C for
15 min showed an abnormal cell elongation, and septum staining indicated that the
septum had not yet formed. However, such morphological behaviour was not
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Yoghurt Science and Technology
observed in autoclaved milk and/or milk heated to 100°C for 15 min (Suzuki et al.,
1986). Furthermore, the presence of sodium formate (40 mg ml-1) in milk induced the
proteolytic activity of L. delbrueckii subsp. bulgaricus so that it became able to
hydrolyse b-Lg, as1- and b-casein compared to only b-casein without the added
formate (Moreira et al., 1997).
Carbon dioxide, which is produced by S. thermophilus (Ascon-Reyes et al., 1995)
had been reported by Driessen et al. (1982) to stimulate the growth of L. delbrueckii
subsp. bulgaricus because part of the CO2 produced by the streptococci disappears
during mixed growth with the lactobacilli. CO2 is produced as a result of urea
hydrolysis and can be measured using an indirect conductance technique (AsconReyes et al., 1995; see also Lanzanova et al., 1993), whilst measuring partial pressure
of dissolved CO2, the concentration of viable cells of the yoghurt micro-organisms
could also be determined (Spinnler et al., 1987). CO2 production in dahi incubated
at 42°C using 1 ml 100 ml-1 starter culture amounted to about 450 ml, and Warsy
(1983) suggested that the gas produced may contribute to the sensory quality of the
product. However, in a recent study, Louaileche et al. (1993, 1996) reported that CO2
and sodium bicarbonate stimulated the growth of S. thermophilus, and exerted a
marked influence on the metabolic activities of the micro-organism, a phenomenon
that has not been reported before.
Other compounds produced by S. thermophilus that stimulate the growth of L.
delbrueckii subsp. bulgaricus are pyruvate and HCO3 (Higashio et al., 1977b, 1978;
Juillard et al., 1987). Other added compounds that stimulated the growth of the lactobacilli are purine, adenine, guanine, uracil and adenosine (Weinmann et al., 1964;
Cogan et al., 1968), monosodium orthophosphate and sodium tripolyphosphate (Yu
and Kim, 1979), oxaloacetic and fumaric acid (Higashio et al., 1977b) and cysteine
at £ 50 mg l-1 (Dave and Shah, 1997). Nevertheless, the action of psychrotrophic bacteria in milk, fortification of the solids of the milk base and/or heating of the milk
can also promote the growth of the yoghurt starter culture (Tramer, 1973; Cousins
and Marth, 1977a, b; Sellars and Babel, 1985; Slocum et al., 1988a, b; for further information refer to Chapter 2).
It can be concluded from the data available, therefore, that the release of stimulatory factors by the yoghurt starter cultures takes place during the incubation
period and, while L. delbrueckii subsp. bulgaricus provides essential nutrients (i.e.
amino acids) for S. thermophilus, the latter produces formate which promotes the
growth of the lactobacilli. Alternatively, the growth characteristics of the yoghurt
organisms can be increased through the application of an electromagnetic field
(Blicq and Murray, 1994) or the use of surface methodology to evaluate some
variables affecting the growth behaviour of the yoghurt organisms (Torriani et al.,
1996).
Amoroso and Manca de Nadra (1990) observed the mutual stimulation in milk,
while in LAPT medium (containing yeast extract, peptone, tryptone and Tween)
with different sugars, only the stimulatory effects of the Streptococcus on the Lactobacillus were observed. This is an expected result as the nitrogen sources in LAPT
medium are readily available and not dependent on proteolytic activity (the mechanism for stimulation of the Streptococcus is the release of peptides by the lactobacilli); thus, the medium used could demonstrate only one side of the partnership.
This underlines the importance of understanding the special qualities of milk as a
growth medium; it has an ample supply of a simple disaccharide and an ample, but
complex source of nitrogen. It is also important to remember that both organisms
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Microbiology of yoghurt and “bio” starter cultures
403
grow perfectly well in milk. Indeed, many of the mild bio-yoghurts are prepared
with mixed cultures, some of which include L. delbrueckii subsp. bulgaricus for a
successful fermentation (see also Marshall and Tamime, 1997).
Although in the 1980s, S. thermophilus was temporarily included as a subspecies
of Streptococcus salivarius (Farrow and Collins, 1984), a separate species was proposed by Schleifer et al. (1991); S. salivarius fails to grow in milk in the presence of
L. delbrueckii subsp. bulgaricus and is not suitable for the manufacture of yoghurt
because of poor flavour, aroma and texture (Marshall et al., 1985). Such observations may also justify the revival of the species, S. thermophilus, even though both
species have similar DNA base compositions and belong in the same DNA homology group. Nevertheless, associative growth was reported between S. thermophilus
and L. helveticus or L. acidophilus (Yoon et al., 1988; Kim et al., 1992) and bifidobacteria stimulated the growth of yoghurt starter cultures (Kumar et al., 1995).
6.3
Factors affecting slow growth of starter cultures
Yoghurt microflora can easily grow in milk and the rate of acid development is faster
due to the growth associated with S. thermophilus and L. delbrueckii subsp. bulgaricus (see Fig. 6.2 and Section 6.2). Nevertheless, the fermentation conditions and
the presence of certain agents or substances in milk may either reduce the rate of
acid development or inhibit growth of the culture and these aspects are summarised
in the following section.
6.3.1 Compounds that are naturally present in milk
There are various antimicrobial systems present in milk and their major role is the
protection of the suckling animal against infection and disease. These inhibitory
systems have been reviewed by Reiter (1978) and their presence in milk can inhibit
the growth of lactic acid bacteria. Auclair and Hirsch (1953) and Auclair and
Berridge (1953) reported the inhibition of starter organisms by raw milk and that
pasteurisation and boiling of the milk improved culture activity. The inhibitory compounds, known as lactenins, are heat sensitive, and are destroyed by heating the milk
to 68–74°C (Auclair, 1954). Patel (1969) reported that S. thermophilus showed a
growth inhibition in fresh raw buffalo’s milk during the first 1–2 hour of incubation,
but a resumptation of growth followed. He proposed that the loss of inhibitory
action was due either to adaptation of the organism to the lactenins or to the
destruction of the lactenins.
Another bactericidal component found naturally in milk is the peroxidase
system, which consists of lactoperoxidase/thiocyanate/hydrogen peroxide [LP/SCN/H2O2 abbreviated as LPS]. Reiter (1978) reported on the sources of these
compounds.
•
•
LP is synthesised in the mammary gland and milk may contain up to 30 mg ml-1
peroxidase which is sufficient to activate the LPS (Reiter, 1985; Nichol et al.,
1995).
SCN- anion is widely distributed in animal secretions and possibly derived
from a rhodanese catalysed reaction with thiosulphate in the liver and kidney;
the SCN- concentration in milk may reach up to 10–15 mg g-1 (Reiter and
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Yoghurt Science and Technology
Härnulv, 1984; Reiter, 1985; Haddadin et al., 1996; see also Prasad and
Sukumaran, 1992).
• H2O2 does not occur naturally in milk (Piard and Desmazeaud, 1991; Nichol et
al., 1995), but its presence in milk is the result of metabolic activity of the lactic
bacteria or from anaerobic growth of other micro-organisms.
In this system, the inhibitory compound is the result of an oxidation reaction where,
in the presence of H2O2, the LP catalyses the oxidation of thiocynate to noninhibitory compounds (SO42-, CO2 and NH3) followed by further oxidation to form
intermediate inhibitory substances, such as hypothiocyanate or higher oxyacids
(Piard and Desmazeaud, 1991; Björck, 1992; Dionysius et al., 1992; Grieve et al.,
1992). However, the inhibition is reversible in the presence of some reducing compounds (e.g. cysteine and dithionite; Reiter, 1978). In general, most starter organisms are resistant to LP systems, but some lactic cultures can give rise to sensitive
mutants (Auclair and Vassal, 1963). Alternatively, continual propagation of starter
cultures in autoclaved milk can affect the susceptibility of the organisms to the LP
system (Jago and Swinbourne, 1959). A preventive measure is the addition of peroxidase to autoclaved milk (Reiter, 1973), or the addition of reducing agents, like
cysteine and dithionite (Reiter, 1978). Incidentally, the LP system is inactivated by
heating milk at 85°C for 16 s (Feagan, 1959a, b), so that heat treatment of yoghurt
milk (85°C for 30 min or 90–95°C for 5–10 min) and the bulk starter milk (93°C for
1–12 –2 hours) are sufficient to destroy the natural inhibitors (Storgards, 1964; Pearce
and Bryce, 1973; Ekstrand et al., 1985; Farkye, 1992). Thus, since H2O2 does not occur
naturally in milk, the mechanism(s) involved in production and inhibition of the
yoghurt organisms is given in detail in Section 6.3.4.
Other inhibitory systems which may warrant some consideration are: (a) bacterial agglutinin which can cause agglutination of the starter organisms, thus affecting their metabolic activity and growth, and (b) certain types of forage, such as
mouldy silage, turnips or vetch, which may result in a milk containing inhibitory substances which can reduce the rate of acid production of the yoghurt starter culture,
even after heating the milk at 90°C for 15 min (see the review by Tamime and Deeth,
1980).
6.3.2 Effect of incubation temperature and inoculation rate
The growth behaviour of S. thermophilus and L. delbrueckii subsp. bulgaricus (i.e.
as single and/or mixed cultures) has been shown in Fig. 6.1 and it is evident that
when the starter culture is incubated at 40–50°C, the optimum rate of acid development is obtained within a very short period. However, in industrial situations,
yoghurt is produced over a short or long period using incubation temperatures at
30°C or 45°C, respectively. In the former method of production, a reduced rate of
acid development becomes inevitable and, although this effect is governed by processing conditions, the quality of the end product could be affected. Some published
data are available and it is recommended that the reader consult the following publications for general information (Tayeb et al., 1984; Mohanan et al., 1984; RadkeMitchell and Sandine, 1986; Jayaram and Gandhi, 1987; Cho-Ah-Ying et al., 1990;
Béal and Corrieu, 1991; Lankes et al., 1998).
The inoculation rate can also affect the rate of acid develoment during the
manufacture of yoghurt. For example, an additional rate of 2–3 ml 100 ml-1 bulk
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Microbiology of yoghurt and “bio” starter cultures
405
starter culture is recommended, whilst a DVI inoculation rate may range between
2.5 and 70 g 100 l-1 depending on the starter culture blend used (i.e. standard or bio
culture). Thus, an inaccurate rate of starter addition to the milk base can affect the
rate of acid development of S. thermophilus and L. delbrueckii subsp. bulgaricus.
6.3.3 Mastitis milk and somatic cell count
Gajdusek and Seleba (1973) reported a 35% reduction in the activity of a yoghurt
culture in milk containing large numbers of somatic cells; however, boiling the milk
for 2 min, or heating to 90°C for 20 min, inactivates the cells completely. Whilst
somatic counts of 4.0 ¥ 105 cells ml-1 cause some inhibition of growth of the yoghurt
organisms with S. thermophilus less resistant than L. delbrueckii subsp. bulgaricus,
complete inhibition of both organisms occurs at counts > 1.0 ¥ 106 cell ml-1 (Mitic et
al., 1982). However, Marshall and Bramley (1984) and Okella-Uma and Marshall
(1986) reported stimulation of S. thermophilus, but inhibition of L. acidophilus,
when these organisms were grown in mastitic milk containing high somatic cell
counts. The stimulation was attributed to increased proteolysis and the inhibition to
increased phagocytic activity of the polymorphonuclear leukocytes. However, Fang
et al. (1993) observed only reduced growth activity of L. acidophilus, L. delbrueckii
subsp. bulgaricus and Lactobacillus paracasei subsp. paracasei in mastitic milk.These
reported differences in the growth behaviour of L. acidophilus could be strain
related.
The quality of yoghurt made from skimmed milk containing a somatic count of
£ 2.5 ¥ 105 cells ml-1 was organoleptically superior to a parallel product made from
milk of ≥ 2.5 ¥ 105 cells ml-1 (Mitchell et al., 1985; Rogers and Mitchell, 1994). Thus,
from the limited data available in this field, it is recommended that yoghurt producers should use milk with a low somatic cell count as reported by Rogers and
Mitchell (1994) (see also Auldist and Hubble, 1998).
6.3.4 Hydrogen perxoide (H2O2)
Hydrogen peroxide is added to raw milk produced in hot countries to improve
its quality during storage. The recommended rate to activate LPS system is 3 mg
100 g-1 of sodium percarbonate (2Na2CO3 ¥ 3H2O2) and 1.4 mg 100 g-1 of sodium
thiocyanate (NaSCN) (IDF, 1988b). However, the natural presence of H2O2 in milk
and activation of LPS, which can inhibit the growth of lactic acid bacteria and other
micro-organisms, is the result of sugar metabolism during fermentation. A wide
range of reactions and catalysing enzymes are involved and these have been recently
reviewed by Condon (1987) and Piard and Desmazeaud (1992).
Oxygen uptake activity and aerobic metabolism of S. thermophilus and L. delbrueckii subsp. bulgaricus have been reported by Smart and Thomas (1987), Teraguchi (1987), Teraguchi et al. (1987) and Condon (1987). H2O2 produced by the
Lactobacillus in the presence of glucose at pH values of 6.5 and 5.0 was apparently
due to the action of cytosolic NADH oxidase (Kot et al., 1996, 1997). Schuts et al.
(1982) reported that the amount of H2O2 (0.8 to 1.8 mg 100 ml-1) produced by L. delbrueckii subsp. bulgaricus was influenced by the strain, growth medium and the type
of added sugars; the highest amount of H2O2 was obtained in UHT milk. However,
lactic acid bacteria can rid themselves of H2O2 formed only by their NADH peroxidase (Piard and Desmazeaud, 1991). The ability of the yoghurt organisms to
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Yoghurt Science and Technology
consume oxygen in milk was about 0.4 mg 100 ml-1 in 24 hour at 25°C (Langeveld
and Bolle, 1985), whilst the influence of dissolved O2 on acid production in buffalo’s
milk by S. thermophilus, L. delbrueckii subsp. bulgaricus and lactococcal species has
been studied by Shekar and Bhat (1983). However, L. acidophilus, S. thermophilus
and some bifidobacterial strains, but not L. delbrueckii subsp. bulgaricus, could
transport Fe2+ into the cell where it is partially oxidised to the ferric form (Kot et
al., 1995); L. delbrueckii subsp. bulgaricus could only oxidise extracellular Fe2+
through the elaboration of H2O2 in the presence of glucose and air.
Therefore, the LPS system can be activated in the presence of H2O2 via two possible routes, the first due to the metabolic activity of the starter cultures and the
second, by the addition of thiocyanate and H2O2. Zall et al. (1983) reported that
when the latter approach was used with rates of 0.2 mM and 0.25 mM, respectively,
it extended the shelf life of raw milk up to 8 days without substantially increasing
the total viable count, but when such milk was used for the manufacture of buttermilk, Cheddar cheese or yoghurt, culture activity was reduced. Nichol et al. (1995)
reported self-induced inhibition of S. thermophilus by activation of LPS, whilst activation of LPS system by adding H2O2 and thiocyanate suppressed acid production
during the manufacturing stages and refrigerated storage of yoghurt (Mehanna and
Hefnawy, 1988; Kumar and Mathur, 1989; Basaga and Dik, 1994; Sarkar and Misra,
1994; Nakada et al., 1996). In a simulated system, L. acidophilus (one strain) and L.
delbrueckii subsp. bulgaricus (three strains) were inhibited in the presence of lactoperoxidase and thiocyanate indicating their ability to produce H2O2 to complete
the LPS system, whilst S. thermophilus, L. helveticus and Lac. lactis subsp. lactis (one
strain) required an external source of H2O2 to cause inhibition by the LPS system
(Guirguis and Hickey, 1987a). The same authors also reported that one strain each
of L. delbrueckii subsp. bulgaricus, L. lactis subsp. lactis and Enterococcus faecium
were resistant to LPS system.
It is evident that the LPS system may inhibit or act as a bacteriostatic agent of
the yoghurt starter cultures. Such effects may possibly depend on the rate of accumulation and/or reduction of the H2O2 (i.e. the activities of NADH oxidase and
NADH peroxidase) in the bacterial cell. Therefore, screening of the yoghurt organisms in relation to the effect of the LPS system may help to overcome production
problems at certain period(s) of the year, stages of lactation, or thiocynate and H2O2
must be used at lower levels than recommended by IDF (1988b).
6.3.5 Antibiotic residues
Antibiotics and/or other antimicrobial agents are used for the treatment of diseases.
One of the major diseases in the dairy cow, which can affect the quality and yield
of milk, is mastitis. Today there are known to be about 1000 different types of antibiotic and the following antimicrobial compounds (penicillin, streptomycin,
neomycin, chloramphenicol, tetracycline, sulphonamide, cloxacillin and ampicillin)
are widely used in the United Kingdom for the treatment of mastitis. The presence
of these antibiotics in milk can either inhibit the growth or reduce the activity of
the yoghurt starter cultures. The sensitivity of these organisms (i.e. single strains or
mixed culture) to these various compounds is shown in Table 6.3 (see also Park et
al., 1984; Sinha, 1984; Hsu et al., 1987; IDF, 1987, 1991a; Herian et al., 1990; Milashki,
1990; Schiffmann et al., 1992; Celik, 1992; Brindani et al., 1994).
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Table 6.3
407
Sensitivity of the yoghurt starter cultures to various antibiotics (ml-1)
Micro-organisms
Antibiotics
Penicillin
Streptomycin
Tetracycline
Chlortetracycline
Oxytetracycline
Bacitracin
Erythromycin
Chloramphenicol
S. thermophilus
L. delbrueckii
subsp. bulgaricus
Mixed culture (IU)
0.004–0.01 IU
0.38 IU
12.5–21.0 mg
0.13–0.5 mg
0.06–1.0 mg
0.4 IU
0.04–0.12 IU
0.3–1.3 mg
0.8–13.0 mg
0.02–0.1 IU
0.38 IU
6.6 mg
0.3–2.0 mg
0.06–1.0 mg
0.7 IU
0.04–0.1 IU
0.7–1.3 mg
0.8–13.0 mg
0.01
1.0
NR
1.0
0.1
0.4
0.04
0.1
0.5
IU, international units; NR, not reported.
Data compiled from Tamime and Deeth (1980), Loussouarn (1983), Schiffmann (1993) and Lim et al.
(1995).
During the intramammary injection of antibiotics for the treatment of mastitis
in the dairy cow, these antimicrobial compounds are retained in the udder tissues
and gradually diffuse into the milk.
Thus, milk from treated cows must be withheld for 72 hours for two main reasons.
First, residual antibiotics in milk are a potential public health hazard and second,
low levels can affect the behaviour and activity of the starter culture (see Table 6.3),
resulting in a poor yoghurt and/or economic loss for the manufacturer. Hence, a
number of governments have introduced a payment penalty scheme for milk containing > 0.004 International Units (IU) of penicillin ml-1; among the test methods
are the disc assay, the 2,3,5-triphenyltetrazolium chloride (TTC), bromocresol
purple (BCP) or the Charm test (see IDF, 1991a and Chapter 10). Some of these
methods use S. thermophilus as the test organism because of its sensitivity to antibiotics (see Table 6.3), but unfortunately the available methods are prone to certain
drawbacks:
•
The sensitivity of S. thermophilus can vary in relation to the strain used (see
Reinbold and Reddy, 1974).
• The above test methods may have certain limitations, for example, Cogan (1972)
observed that L. delbrueckii subsp. bulgaricus is more sensitive than S. thermophilus to streptomycin, and to cause a 50% inhibition of growth, 1.6–4.45 and
7.3–13.00 mg ml-1 of streptomycin were required, respectively. Thus, a milk which
passes the antibiotic test may contain enough streptomycin to inhibit the growth
of L. delbrueckii subsp. bulgaricus (see also Park et al., 1984). However, comparative growth of S. thermophilus and L. delbrueckii subsp. bulgaricus in milk
containing streptomycin showed that the latter micro-organism was more sensitive (Ramakrishna et al. (1985); again strain differences appear to be important.
The major effect of antibiotic residues in yoghurt milk is to cause a breakdown
in the associative growth between S. thermophilus and L. delbrueckii subsp. bulgaricus, or a slow down in the rate of acid development (i.e. longer processing time)
YOG6 6/1/99 5:26 PM Page 408
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Yoghurt Science and Technology
and this can, in turn, lead to syneresis or wheying-off. To combat such problems, the
following measures have been recommended:
•
•
The use of milk for the manufacture of yoghurt that is free from antibiotics.
The addition of penicillinase or penicillinase-producing organisms, e.g. Micrococcus spp., to milk in order to inactivate residual penicillin contamination
(Reiter et al., 1961; Vazquez and Reiter, 1962).
• Heat treatment of milk can reduce the potency of some antibiotics. Tramer
(1973) reported an 8% inactivation of penicillin at 72°C for 15 s, or 20% at 87.7°C
for 30 min, or 50% at commercial sterilisation temperatures; tetramycin lost 2/3
of its potency at 85°C for 30 min, but streptomycin and chloramphenicol
remained stable and unaffected.
• Lowering the water activity of the growth medium with glycerol for S. thermophilus (Aw from 0.992 to 0.995) and L. delbrueckii subsp. bulgaricus (Aw from
0.992 to 0.985) improved the resistance of these organisms against penicillin, but
not gentamycin (Larsen and Anon, 1989b).
It is most likely that the inhibitory effect on these organisms is influenced by the
mode of action of the antibiotics and, in view of the immense number of antimicrobial drugs used in veterinary medicine, an attempt has been made to classify
only the most widely used antibiotics. The overall characteristics of this group and
their possible effect on the yoghurt starter cultures is shown in Table 6.4. Furthermore, depending on the type of antibiotic used, the mode of action of these drugs
on S. thermophilus and L. delbrueckii subsp. bulgaricus can be summarised as
follows: (a) interference with the cell membrane structure and permeability, (b)
interference with cellular metabolism of proteins, carbohydrates and lipids, (c) interference with energy-yielding transformations in the cell, (d) inhibition of various
enzymes and phosphorylation systems, and (e) blocking the synthesis of DNA and
RNA during cell division.
Antibiotic-resistant yoghurt strains (see Table 6.5) have been induced to resist
higher concentrations of antibiotics by repeated subculturing in milk containing
varying concentrations of the antibiotics (Babu et al., 1989a; see also Yondem et al.,
1989; Bozoglu et al., 1996). However, the quality of yoghurt produced by such strains
was not reported, but Babu et al. (1989a) reported the penicillin-resistant L. delbrueckii subsp. bulgaricus showed almost 50% reduction in acetaldehyde production, whilst the streptomycin-resistant cultures exhibited appreciable depression in
flavour production. Thus, these developed cultures may have different characteristics, such as reduced rates of acid and flavour production, or the inability to ferment
certain carbohydrates, and these changes could adversely affect the performance of
a culture during commercial production (see Babu et al., 1989a, b; Chirica et al.,
1998). Furthermore, genes for drug-resistance play an important role as genetic
markers, and spontaneous frequencies of mutation to antibiotic resistance interfere
with genetic research for the improvement of starter cultures for fermentation
(Curragh and Collins, 1992).
6.3.6 Detergent and disinfectant residues
Detergents and disinfectants are widely used in the dairy industry for cleaning and
sanitising dairy equipment on the farm and in the creamery (see Chapter 4). The
general specification and classification of these preparations is discussed elsewhere,
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Microbiology of yoghurt and “bio” starter cultures
Classification and mode of action of some antibiotics
Source or origin
Microbial
Streptomyces spp.
Antibiotics
produced
Streptomycin
Production
(%)
Ë
Table 6.4
409
Tetracyclines
Neomycin
Ì
Erythromycin
58
Chloramphenicol
Sulphonamide
Ë
Penicillin
Chloramphenicol
Ì
Drugs extracted
from algae, lichens
and animals
Reaction or site inhibited
is folate synthesis
* Cell wall inhibitors
** Protein synthesis
inhibitors
3
Ê
Miscellaneous
Ì
Alkaloids
12
* Cell wall inhibitors
* Alter cell membrane
permeability
* Disorganise cell
membrane structure
* Disorganise cell
membrane structure
Ê
Plant extracts
Nucleic acid inhibitors
Ë
Synthetic
Polymyxin
9
Ê
Bacillus polymyxa
Ì
Tyrocidin
18
Ê
Fusidium coccineum
Aspergillus fumigatus
Bacillus licheniformis
Bacillus brevis
* Protein synthesis
inhibitors
** Protein synthesis
inhibitors
* Protein synthesis
inhibitors
* Protein synthesis
inhibitors
** Protein synthesis
inhibitors
* Cell wall inhibitors
* Protein synthesis
inhibitor
* Cell wall inhibitors
Ë
Penicillin
Xanthocillin
Fusidic acid
Fumagillin
Bacitracin
Gramicidins
Ì
Penicillin notatum
Ë
Ristocetin
Gentamicin
Ê
Nocardia spp.
Micromonospora spp.
Possible function and
mode of action on the
yoghurt starter culture
* Bactericidal. ** Bacteriostatic.
Adapted from Garrod et al. (1973) and Edwards (1980).
but basically, the detergent formulations contain alkali compounds (e.g. sodium
hydroxide), while the sanitising agents are quaternary ammonium compounds
(QAC) or iodine or chlorine-based compounds.
Inorganic acids are also used for cleaning and disinfecting purposes. Therefore,
residues of these compounds in milk can be attributed to two main causes. First negligence, bad management or a faulty cleaning-in-place (CIP) system (i.e. on the farm
or at the factory); the latter is more likely to occur on the farm or in milk tankers.
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Table 6.5
Development of yoghurt starter cultures resistant to different antibiotics
Antibiotics
Penicillin
Streptomycin
Achieved resistance (ml-1)
References
3 IU
500 mg
Hargrove et al. (1950)
Chlortetracycline
Chloramphenicol
Streptomycin
70–120 mg
40–50 mg
500 mg
Solomon et al. (1966)
Ampicillin
Cephalexin
Chlortetracycline
50 mg
150 mg
£ 50–150 mg
Ferri et al. (1979)
Penicillin
Streptomycin
0.25 IU
500 mg
Babu et al. (1989a, b)
Table 6.6 Sensitivity of the yoghurt starter cultures to various detergent disinfectants and
pesticides (mg l-1)
Micro-organisms
Inhibitory
substances
Disinfectant/detergent
Chlorine compounds
QAC
Ampholyte
Iodophore
Alkaline detergent
Insecticides
Malathion
N-methylcarbanate
S. thermophilus
L. delbrueckii
subsp. bulgaricus
5–100
100–500
2.5–100
0.5–100
10–60
60
Mixed culture
50–> 2500
> 250
> 1000
> 2000
500–1000
200
20
Adapted from Tamime and Deeth (1980), Guirguis and Hickey (1987b) and Petrova (1990).
Second, it is the practice of some milk producers overseas to add biocidal compounds (e.g. H2O2) to milk in order to improve its keeping quality. This latter
approach is not recommended for public health reasons and the presence of such
compounds in milk can adversly affect, or totally inhibit, the growth of starter
cultures.
It can be observed from Table 6.6 that the susceptibility of S. thermophilus and
L. debrueckii subsp. bulgaricus to cleaning residues is increased in monocultures
compared with mixed cultures and this variation could be attributed to:
•
differences or variations in the strains of bacteria being used by different
researchers (Liewen and Marth, 1984; Guirguis and Hickey, 1987b; El-Zayat,
1987; Mäkelä et al., 1991);
• variation between batches of the commercial detergents and disinfectants tested;
• variation in the test method used to measure the levels of inhibition (see
Lanzanova et al. (1991) for the use of a conductimetry technique to evaluate
the effects of disinfectants and detergents on the activity of starter cultures);
• greater resistance as a result of associative growth relationships.
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Microbiology of yoghurt and “bio” starter cultures
411
Another possible source of detergent and/or sterilant residues is the glass bottle
washer, for in some countries, glass bottles are still used for packaging stirred or set
yoghurt. In the latter type of yoghurt, Nikolov (1975) concluded that if the milk contained above 2.5% of bottlewash liquid, consisting of 1% sodium hydroxide and
hypochlorite (i.e. the chlorine concentration > 100 mg l-1), the concentration was
high enough to inhibit the growth of S. thermophilus and L. delbrueckii subsp.
bulgaricus.
6.3.7 Environmental pollution
Incidents of insecticide residues in milk have been reported and this occurrence
could well be due either to post-milking contamination, or to feeding cattle with
fodder that has been sprayed with an insecticide to combat disease. Milk containing malathion (200 mg l-1) or N-methylcarbamate (20 mg l-1) will inhibit the growth
of the yoghurt organisms (see Table 6.6). However, Deane and van Patten (1971)
observed that 100 mg l-1 of malathion or trichlorphon in milk had little effect on the
rate of lactic acid development by yoghurt cultures, but some variation in cell morphology did occur after several culture transfers. When viewed under a light microscope (using ordinary staining techniques) the recorded changes included a
decrease or increase in cell size and the formation of longer chains. In addition,
Deane and Jenkins (1971) propagated L. delbrueckii subsp. bulgaricus alone in milk
containing the same insecticides and observed various morphological changes under
the electron microscope. The rod cells were longer, wider or narrower and showed
a compact protoplasm and frequent flaking of the cell wall material, and there were
fewer cross-walls produced.
In the 1980s, Egyptian scientists intensified their research into the fate of different pesticides (e.g. aldicarb, chlorpyrifos, deltamethrin, lindane, fenvalerate
(pyrethroid), malathion and DDT) during the manufacture of zabadi and cheese,
and on the growth behaviour of starter cultures (Shaker et al., 1985, 1988; Ismail et
al., 1987; Magdoub et al., 1989; Zidan et al., 1990; see also Misra et al., 1996). The
results of these studies could be summarised as follows:
•
•
•
•
•
The pesticide concentration decreased in freshly made zabadi.
Gelation time of the milk increased and the cheeses had many holes.
Cells of L. delbrueckii subsp. bulgaricus floculated into clumps in milk containing aldicarb and the cell count was lower than the control.
Heating of the pesticide-contaminated milk and fermentation contributed
towards the degradation of pesticides.
Reduced growth rates of S. thermophilus in the presence of fenvalerate or DDT
were observed, whilst L. delbrueckii subsp. bulgaricus was sensitive to malathion
and DDT.
6.3.8 Bacteriophages
Bacteriophages (phages) are viruses which can attack and destroy the yoghurt
organisms and the resultant failure of lactic acid production leads to poor coagulation of the process milk. The occurrence of such viruses in mesophilic dairy starter
cultures (e.g. cheese starters) was first reported by Whitehead and Cox (1935) and,
for the past few decades, research work on the phages of mesophilic lactic acid
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Yoghurt Science and Technology
bacteria has been intensified, primarily because of the economic importance of
cheese in the dairy industry. However, interest in bacteriophages that can attack
thermophilic lactic acid bacteria (i.e. the yoghurt cultures) has been aroused first
because world production figures of yoghurt have increased significantly and
product failure results in great economic loss to the industry; second because the
manufacture of yoghurt is more centralised and bacteriophage attack could become
a major problem; and third because strains of S. thermophilus and L. delbrueckii
subsp. bulgaricus are widely used in the manufacture of high temperature scalded
cheese (e.g. the Swiss varieties) and hence bacteriophage problems could result in
both a slow “make” and a low quality cheese. As a consequence, research work on
bacteriophages has intensified and a large number of publications are available.
However, some selected reviews on bacteriophages of S. thermophilus and L. delbrueckii subsp. bulgaricus are recommended for further information (Reinbold and
Reddy, 1973; Sozzi et al., 1981; Stadhouders et al., 1984; Thunell and Sandine, 1985;
Ackermann and DuBow, 1987; Mata and Ritzenthaler, 1988; Sechaud et al., 1988;
Rajagopal and Sandine, 1989; Jarvis, 1989; Cogan and Accolas, 1990; Coffey et al.,
1994; Sable and Lortal, 1995; Gasson, 1996; Neve, 1996; Auvray et al., 1997;
Josephsen and Neve, 1998).
The general morphology of a bacteriophage consists of a head and protruding
tail, and the type capable of infecting lactic acid bacteria may consist of a double
strand of DNA in a linear form which is located in the head (Lawrence et al., 1976;
Sandine, 1979; Neve, 1996). The guanine plus cystine (G + C) content of the bacteriophage is somewhat similar to the G + C composition of the bacterial hosts’
chromosomes; thus in principle, such similarity may explain the close relationship
between the bacteriophage and the host. Over the years different methods have
been proposed to classify bacteriophages (Pette and Kooy, 1952; Bradley, 1967;
Lawrence et al., 1976; Soldal and Langsrud, 1978; Koroleva et al., 1978; Mullan, 1979),
but they were not accepted universally. However, a recent approach to bacteriophage taxonomy, which is accepted universally, has identified three groups known
as bacteriophage families, namely the Myoviridae, Podoviridae and Siphoviridae
(Ackermann and DuBow, 1987; Francki et al., 1991). Bacteriophages of S. thermophilus and L. delbrueckii subsp. bulgaricus belong to the Siphoviridae family
(Neve, 1996; Josephsen and Neve, 1998), and Fig. 6.3 illustrates an example of an
isometric head structure of a bacteriophage of S. thermophilus. The overall morphology of bacteriophages of the yoghurt starter cultures are described as having
an isometric head with non-contractile tails. Some bacteriophages may have a collar
situated under the head and a base plate at the terminal tail structure including
spikes (see Soldal and Langsrud, 1978). Bacteriophages are classified into two main
categories depending on the growth responses in the bacterial host, and these types
are virulant or lytic bacteriophages (i.e. those that can infect and lyse the host cell)
and temperate, prophage or lysogenic bacteriophages (i.e. those that do not lyse the
bacterial host, but instead insert their genome in the host chromosome) (Neve,
1996).
The lytic cycle of a bacteriophages involves several stages known as adsorption
to the bacterial host, injection of bacteriophage DNA, bacteriophage maturation
and lysis of the bacterial cell. The lysogenic cycle primarily involves only the first
two stages since rather than the bacteriophage maturing in the bacterial host, the
bacteriophage DNA is inserted into the bacterial chromosome. According to Neve
(1996) and Josephsen and Neve (1998), this action occurs by a single reciprocal
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413
Fig. 6.3 Illustration of a transmission electron micrograph showing different
morphological characteristics of a virulent bacteriophage of S. thermophilus with (A) and
without (B) a tail fibre
Reproduced with permission of H. Neve.
recombination event taking place at a specific region of homology between the bacteriophage DNA and the bacterial host DNA which is known as an attachment site
(i.e. attP in the bacteriophage genome and attB in the bacterial host). Thus, bacterial host lysis does not occur and the bacteriophage DNA is now known as probacteriophage and is replicated simultaneously with bacterial host DNA, giving rise to
a progeny of lysogenic cells. This bacteriophage is known as a temperate bacteriophage. Over the years, many researchers have used electron microscopy to observe
the morphology of bacteriophages of S. thermophilus and L. delbrueckii subsp. bulgaricus (see Table 6.7).
Accolas and Spillmann (1979a) observed that six out of seven S. thermophilus
bacteriophages were similar, that is the head, which was polyhedral or possibly octahedral, was 49–53 nm in diameter, the tail length ranged from 200 to 224 nm (with
the exception of one, i.e. 130 nm) and the tail width from 8 to 9 nm; the tail tip had
a small plate covered with short prongs or a fibrous mass; the seventh type of phage
had no specific tail-tip structure. However, a recent study by Krusch et al. (1987)
suggested that streptococcal bacteriophages obtained from different research laboratories in Europe have different morphological sizes (see Table 6.7). The distinctive characteristics of S. thermophilus bacteriophages can be summarised as
follows:
•
The sensitivity of the organism to bacteriophage attack was described by Pette
and Kooy (1952) under one of three headings: bacteriophage-insensitive,
bacteriophage-tolerant (i.e. carriers of the particles) and bacteriophage-sensitive
(i.e. results in complete lysis of the host cell).
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Table 6.7
Morphology (range) of bacteriophages of yoghurt starter cultures
Head
Tail
Micro-organism
a
Length (nm) ¥ diameter (nm)
Structure
Size (nm)
S. thermophilus
Hexagonal
Polyhedron
Polyhedral or octahedron
NR
Polyhedral
Isometric
Hexagonal
Isometric
50–60
40–60
49–50
60–65
48–70
57
45–65
65
217–239 ¥ 4.8
220–420 ¥ 8
130–224 ¥ 8–9
236–290 ¥ 10
213–265 ¥ 11–12
234 ¥ 9.5 (mean)
220–245 ¥ NR
230–260 ¥ NR
L. delbrueckii
subsp. bulgaricus
Hexagonal
Polyhedral or octahedron
NR
Hexagonal
56–62
44–55
50–59.4
47
205–215 ¥ NR
116–160 ¥ 8–9
175–198 ¥ 5–6.6
159 ¥ NR
n = Number of strains tested. NR: not reported.
Tail
Tip
(n =)b
References
+
+
±
+
+
+
2
2
14
3
59
50
120
24
Sarimo and Moksunen (1978)
Koroleva et al. (1978)
Accolas and Spillmann (1979a)
Reinbold et al. (1982)
Krusch et al. (1987)
Carminati et al. (1994)
Fayard et al. (1993)
Brüssow et al. (1994)
+
±
-
1
7
3
1
Peake and Stanley (1978)
Accolas and Spillmann (1979b)
Reinbold et al. (1982)
Auad et al. (1997)
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Microbiology of yoghurt and “bio” starter cultures
415
•
A similar classification was proposed by Sarimo and Moksunen (1978), but they
incorporated some morphological features as well. Russian workers (Koroleva
et al., 1978) divided the bacteriophages of S. thermophilus into two groups based
on morphological observations: regular polyhedron head 40 nm in diameter and
others, i.e. head size 65 nm in diameter.
• All virulent bacteriophages of S. thermophilus belong to one DNA homology
group (e.g. genome size 37–44 kb (Kivi et al., 1987; Neve et al., 1989; Larbi et al.,
1990, 1992; Fayard et al., 1993; le Marrec et al., 1997) and based on the protein
profiles and degree of homology of these bacteriophages, they were classified
into two or three subgroups (see also Prevots et al., 1989; Benbadis et al., 1990;
Sebastiani and Jäger 1992, 1993; Brüssow et al., 1994; Bruttin et al., 1997a, b).
• Larbi et al. (1992) identified three different mechanisms of bacteriophage resistance in the bacterial host, one of which exhibited a temperature-dependent
response.
• Expression of a Lac. lactis subsp. lactis plasmid-encoded bacteriophage defence
mechanism in S. thermophilus increased the bacteriophage resistance in the
Streptococcus (Moineau et al., 1995).
• The conductance measurement technique and spot test method have been used
successfully for bacteriophage detection in S. thermophilus and a yoghurt
culture, respectively (Carminati and Neviani, 1991; Champagne and Gardner,
1995).
• Lysogenic strains and many temperate bacteriophages of S. thermophilus may
have an endogenous origin (Carminati and Giraffa, 1992).
Virulent bacteriophages attacking S. thermophilus host cells result in lysis of the cell
wall by an enzyme, lysin, which releases newly formed bacteriophages into the
growth medium. A typical illustration of which can happen to such a culture before
and after infection with a bacteriophage is shown in Fig. 6.4.
In the 1970s, some of the distinctive morphological features of L. delbrueckii
subsp. bulgaricus bacteriophages were reported by Peake and Stanley (1978) and
Accolas and Spillman (1979b) and, in brief, they are (a) shorter in overall length in
comparison with S. thermophilus bacteriophages (e.g. 116–198 nm) with the exception of those phages studied by Peake and Stanley (1978), where the length varied
from 205 to 215 nm, (b) the presence of a “collar” structure, and (c) the appearance
of up to ten “cross-bar” structures intersecting the tail at intervals (see Table 6.7).
More recent characterisations of the lactobacillar bacteriophages have included the
following:
•
Both lytic and temperate bacteriophages have been found in L. delbrueckii
subsp. bulgaricus and subsp. lactis, and their classification has been reported by
Cluzel et al. (1987a, b), Sechaud et al. (1988) and Lahbib-Mansais et al. (1988).
• A temperate bacteriophage infecting L. delbreuckii subsp. bulgaricus had a circularly permuted and terminally redundant genome with a unique sequencing
of 36 kb, and was capable of infecting L. delbrueckii subsp. lactis (Boizet et al.,
1990; Lahbib-Mansais et al., 1992; Auad et al., 1997); a virulent bacteriophage has
a linear genome of 35 kb (Chow et al., 1988).
• Vescovo et al. (1990) reported on the sensitivity to bacteriophages of morphological variants of L. delbrueckii subsp. bulgaricus (e.g. curved or straight cells)
and suggested that the physiological reactions were influenced by calcium and
magnesium.
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Yoghurt Science and Technology
Fig. 6.4 Scanning electron micrograph illustrating (A) a healthy S. thermophilus culture
and (B) the lysis of cells after infection with a virulent bacteriophage
Reproduced with permission of H. Neve.
No data are available on infection of L. acidophilus and Bifidobacterium species
with bacteriophages. It could be argued that the presence of these cultures in bioyoghurt is for the provision of probiotic cells in the product rather than for the production of acid for the gellation of milk. However, the interest generated in using
these organisms as starter cultures may initiate research work on their bacteriophages. Research work on the bacteriophages of thermophilic lactic starters has
increased substantially since the 1970s and Table 6.7 reviews the morphology of
those bacteriophages of the yoghurt organisms that have been reported in the literature up to the present time. Figure 6.5 shows some of the morphological characteristics of the bacteriophages that can infect L. delbrueckii subsp. bulgaricus.
It is also relevant, concerning the viruses attacking S. thermophilus and L. delbrueckii subsp. bulgaricus, that:
•
•
If milk is the origin of bacteriophage contamination, then heat treatment at 85°C
for 20 min ensures their destruction (Stolk, 1955); raw milk could be the source
of bacteriophages, thus causing problems during the manufacture of some traditional cheeses from raw milk in Europe.
The optimum temperature of bacteriophage proliferation is the same as the
optimum growth temperature of the host, i.e. S. thermophilus phages at 39–40°C
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417
Fig. 6.5 Electron micrograph showing the morphology of two bacteriophages of L.
delbrueckii subsp. bulgaricus
(A) Bacteriophage c5 of strain LT4; note the isometric head, non-contractile flexible and regularly
striated tail and large basal plate. (B) Bacteriophage y5 of strain Y5; note the isometric head with
large triangular “facets”, a small fibrous collar and tail tip composed of short fibres.
Reproduced with permission of J.-P. Accolas and H. Spillman.
and L. delbrueckii subsp. bulgaricus bacteriophages at 42–43°C (Sozzi et al.,
1978).
• Chemical sterilisation of equipment using 0.1% QAC, 70–90% ethanol,
0.5–1.0% potassium permanganate or 50–100 mg l-1 of available chlorine causes
the destruction of S. thermophilus phages (Ciblis, 1966); peracetic acid (120–
300 mg l-1) and active chlorine (≥ 2.6 mg l-1) were recommended by Langeveld
and van Montfort-Quasig (1995, 1996) for inactivating yoghurt starter culture
bacteriophages (see also Neve et al., 1996).
• Phages are species and/or strain specific, i.e. phages of mesophilic lactic starters
do not attack thermophilic starter cultures, and furthermore, S. thermophilus
phages do not attack L. delbrueckii subsp. bulgaricus strains.
• The lysis of Lactobactillus species including L. delbrueckii subsp. bulgaricus in
the vagina was due to the action of bacteriocins produced by certain lactobacilli
and bacteriophages (Tao et al., 1997).
It is evident, therefore, that one or more of the following precautionary measures
should be practised in order to eliminate or control phage attack (see also IDF,
1991b):
•
•
•
•
•
•
•
•
use of aseptic techniques for the propagation and production of starter cultures;
ensure effective sterilisation of utensils and equipment;
ensure proper heat treatment of the milk;
restrict movement of plant personnel in starter handling room, and locate starter
room far away from production area;
check filtration of air into the starter room and production area;
“fog” the atmosphere in the starter room with hypochlorite solution (not to be
encouraged) or use laminar-flow cabinets for small-scale culture transfers;
grow starter culture in bacteriophage inhibitory medium (BIM);
use a daily rotation of bacteriophage unrelated strains (or phage-resistant
strains) of S. thermophilus and L. delbrueckii subsp. bulgaricus (Havlova and
Jicinska, 1985);
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Yoghurt Science and Technology
•
produce the bulk starter culture or even the retail product using a direct-to-vat
system;
• the use of turpine (obtained from aromatic plants by steam distillation) at a rate
of 500 mg 100 l-1 or black pepper oil inhibited bacteriophage infection but not the
growth of L. delbrueckii subsp. bulgaricus (Wolf et al., 1983);
• growth of the yoghurt organisms in soy-milk stopped bacteriophage infection
(Farhat et al., 1984).
6.3.9 Bacteriocins
Antibacterial substances (usually segregated from antibiotics) are produced by a
wide range of bacteria including dairy starter cultures. They were termed colicinlike, but currently they are known as bacteriocins. For further information refer to
the following reviews (Piard and Deamazeaud, 1992; Nettles and Barefoot, 1993;
Barefoot and Nettles, 1993; Hoover and Steenson, 1993; de Vuyst and Vandamme,
1994; Nes et al., 1996; Marshall and Tamime, 1997). In general, Tagg et al. (1976) characterised bacteriocins as follows:
•
•
•
•
•
proteinaceous in nature
bactericidal rather than just bacteristatic
capable of linking to specific binding sites on the bacterial cells and showing different activity from other antimicrobial substances
plasmid-mediated
Active against bacteria of the same genera.
At present, around 70 different types of bacteriocins have been identified and produced by lactic acid bacteria. Table 6.8 summarises some selected characteristics of
the bacteriocins produced by S. thermophilus and L. delbrueckii subsp. bulgaricus,
and careful selection of the streptococci strains of the starter culture blend is important to minimise their inhibition. However, other lactic acid bacteria including
Propionibacterium species can produce bacteriocins that are slightly inhibitory to
L. delbrueckii subsp. bulgaricus (see Table 6.9). The use of such organisms beside
the yoghurt starter culture is aimed at controlling over- or postacidification in the
product (Weinbrenner et al., 1997). It could be of practical relevance that a bacteriocin produced by S. thermophilus affected the growth of L. delbrueckii subsp.
bulgaricus only in M17 broth and not in milk (Cilano et al., 1991; see also Sikes and
Hilton, 1987).
Limited data have been published on the mode of action of bacteriocins produced by lactic acid bacteria that can affect the yoghurt starter cultures. For
example, lacticin B is bactericidal to sensitive cells, but it does not cause cellular
lysis of host cells. It adsorbs non-specifically to sensitive and insensitive lactobacilli
because it is a highly hydrophobic peptide and the mode of action may be similar
to nisin and pediocin AcH (de Vuyst and Vandamme, 1994).
6.3.10
Miscellaneous factors
6.3.10.1 UF milk
The associative growth by S. thermophilus and L. delbrueckii subsp. bulgaricus
was lower in ultrafiltered (UF) milk than in milk (Tayfour et al., 1981). A similar
Some selected characteristics of bacteriocins produced by yoghurt starter cultures
Starter organisms
and strain
Bacteriocin name
Molecular
mass (kDa)
NR
< 0.7
NR
STB 40 and 78
10–20
Lipase, a-chymotrypsin,
trypsin and pronase
ST 10
ST 10
> 100
SFI 13
Thermophilin 13
4.0
Proteolytic enzymes
and a-amylase
NR
Bulgarican
NR
NR
7994
NR
< 0.7
NR
CFR 2028
NR
NR
NR
S. thermophilus
STB 40 and 78
L. delbrueckii subsp.
bulgaricus DDS 14
Sensitivity
Comments
Antimicrobial compound is heat stable (100°C for 10 min) and
and displayed inhibitory activity to Gram-negative and
Gram-positive bacteria.
Both bacteriocins are stable between pH 2 and 12 and are
heat resistant; they are active against Enterococcus spp. and
S. thermophilus strains.
Only active against S. thermophilus and heat stable at 121°C
for 15 min.
Thermophilin is heat stable (100°C for 1 hour) and active in
the pH range 1.6–10.
Thermostable (120°C for 60 min) and only active at acidic pH;
displayed a wide spectrum of inhibiting Gram-positive and
Gram-negative bacteria.
Still active at pH 4 and thermostable for 1 hour at 100°C; it is
active against Pseudonomas and Staphylococcus species.
Active principal of the bacteriocin is proteinaceous in nature;
stable at pH 3.8–5.0 and heat for 75°C for 30 min; active
against Bacillus cereus.
NR, not reported.
Data compiled from Abdel-Bar et al. (1987), Cilano et al. (1990, 1991), Marshall and Tamime (1997) and Balasubramanyam and Varadaraj (1998).
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Table 6.8
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Table 6.9 Inhibition of yoghurt starter cultures by bacteriocins produced by different microorganisms
Micro-organisms
Bacteriocin name/molecular
mass (kDa)
L. delbrueckii subsp.
lactis
Lacticin A & B
kDa (NR)
Lac. lactis subsp. lactis
L. acidophilus
L. helveticus
Propionibacterium
jensenii
Lactobacillus reuteri
Lac. lactis subsp. lactis
Lactobacillin G4
kDa (NR)
No name
< 1 kDa
No name
1–10 kDa
Lacticin 481
1.3–2.9 kDa
Acidophilucin A
kDa (NR)
Lactacin B
6.2–8.1 kDa
Lactacin F
2.5–6.3 kDa
Helveticin J
37 kDa
Jenseniin G
Reutericin 6
2.7 kDa
Lactococcin DR
2.3–2.4 kDa
Lacticin 481
1.3–2.9 kDa
Comments and references
Inhibited growth of L.
delbrueckii subsp. bulgaricusa
(Toba et al., 1991)
As above (Giraffa et al. 1989,
1990)
As above (Hara et al., 1995)
As above (Su and Lin, 1990)
As above (Piard and Desmazeaud,
1992)
As above (de Vuyst and
Vandamme, 1994)
As above (Weinbrenner et al.,
1997)
As above (Kabuki et al., 1996)
Inhibited growth of S.
thermophilus (de Vuyst and
Vandamme, 1994)
NR, not reported.
a
Lacticin A is active against this micro-organism.
observation was recently reported by Radulovic and Obradovic (1997), but they
observed that the lactobacilli showed better acid development than the streptococci.
Ozen and Ozilgen (1992) reported that the kinetic analysis clearly illustrated that
the contribution of each microbial species of the yoghurt organisms to the mixed
culture growth changed drastically when the substrate concentration was about
15 g 100 g-1.
6.3.10.2 Added flavours
The addition of coffee (Coffee robusta) extract, ginseng saponins and garlic extract
to the milk base before fermentation reduced acid development during the manufacture of yoghurt, dahi and acidophilus milk, or in milk inoculated with single
strains of lactic acid bacteria (Kim et al., 1987; Gandhi and Ghodekar, 1988; Fardiaz,
1995).
6.3.10.3 Lysozyme
This compound is sometimes added to cheese milk to control or inhibit the growth
of clostridia. Most of the L. helveticus strains have been found to be sensitive to
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421
lysozyme at low concentrations of 10 or 20 mg ml-1, but not the yoghurt starter organisms (Neviani et al., 1988a). However, a strain of L. delbrueckii subsp. bulgaricus that
was sensitive to lysozyme was cultured eight times in the presence of 100 mg g-1 of
lysozyme; it developed some resistance but lost it on subsequent culturing in milk
(Neviani et al., 1988b); lysozyme resistance is thought to be plasmid related (see also
Mercenier et al., 1988a, b).
6.3.10.4 Diet of the cow
At certain times of the year (i.e. June to August in Italy), the acidification rate of
the yoghurt organisms is reduced, but activity is retained when the milk is supplemented with paraffin, vitamin E or Fe2+ and Zn2+; the problem may also be reduced
by supplementing the cow’s diet with vitamins (Maianti et al., 1996).
6.3.10.5 Nitrates (NaNO3) and nitrites (NaNO2)
The presence of nitrites in some dairy products is permitted at a level of 0.01 mg
100 ml-1 (Baranova et al., 1997). However, the addition of the nitrates or nitrites to
the milk base reduced the rate of acid development by yoghurt cultures
(Korenekova et al., 1997; Baranova et al., 1997) and the resulting products had low
viscosities. Changes in the NaNO3 content in yoghurt, including interactions with
caseins, have been reported by Steinka and Przyblowski (1994, 1997).
6.3.10.6 Radioactive materials (131I)
Contamination of milk with such components is undesirable, but in view of the
Chernobyl accident, Greek scientists studied the effect of adding 131I to milk during
the manufacture of yoghurt and labneh (Vosniakos et al., 1991, 1992, 1993; see also
Section 5.7 in Chapter 5; Micic et al., 1985). An 131I content in milk amounting to
6–12 kBq kg-1 reduced the counts of S. thermophilus and L. delbrueckii subsp.
bulgaricus by 45–52% in set yoghurt and labneh; lactococcal species were reduced
by 30% in cheese and buttermilk and 26% in ripened butter.
6.3.10.7 Aflatoxins
Aspergillus flavus and parasiticus have been identified as producing toxins (AFB1&2
and AFG1&2) that have been implicated as acute toxicants and heptacarcinogens in
the human (El-Nezami and Ahokas, 1998). Their presence in yoghurt is discussed
in Chapter 10, but research work regarding the role of lactic acid bacteria in
controlling the growth of Aspergillus species is very limited. However, certain
mesophilic and thermophilic starter cultures are capable of detoxifying aflatoxin
(El-Nezami and Ahokas, 1998).
Mohran et al. (1985) showed that whereas AFB, added to skimmed milk (up to
0.44 mg ml-1), did not affect the growth of S. thermophilus and lactococcal species,
L. delbrueckii subsp. bulgaricus and L. paracasei subsp. paracasei were inhibited, but
Kalra et al. (1977) observed the opposite effect on the yoghurt organisms; the
yoghurt starter cultures were very effective in the detoxification of 0.5 mg l-1 ochratoxin A present in milk (Rasic et al., 1991; Skrinjar et al., 1996).
6.3.10.8 Sweetening agents
The addition of sugar ≥ 9 g 100 g-1 to the milk may cause inhibition or delay in the
fermentation period, as will the addition of artificial sweetners. For further details
refer to section 2.6 in Chapter 2 (see also Lacroix and Lachance, 1988a, b, 1990;
Larsen and Anon, 1989a, 1990; Latrille et al., 1992).
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Yoghurt Science and Technology
6.3.10.9 Cadmium (Cd)
As a result of environmental pollution, Cd may be found in cow’s milk at low levels
up to 160 mg kg-1 with typical values < 0.5 mg kg-1 (Walstra and Jenness, 1984).An inhibition of the decrease in pH was observed for S. thermophilus > 5 mg Cd l-1 (Korkeala
et al., 1984), but not at lower levels.
6.3.10.10 Phosphates
Bacteriophage inhibitory media (BIM) for lactococci contain high levels of phosphates which chemically bind the free calcium in milk, thus preventing bacteriophage replication (Zottola and Marth, 1966). However, the growth of L. delbrueckii
subsp. bulgaricus in phosphated milk (i.e. added phosphate or commercially available BIM) was inhibited and cellular morphology was altered in milk containing
about 3 g 100 g-1 phosphate (Wright and Klaenhammer, 1983, 1984). Shalaby et al.
(1986) observed no effect on growth of four strains of S. thermophilus in phosphated
media and when milk containing sodium citrate + sodium phosphate, yeast extract
and infected with bacteriophage was used, the rate of acid production was not
reduced either; the presence of the buffering agents was effective in suppressing
bacteriophage attack. However, Champagne and Gange (1987) observed that the
starter activity of three strains of S. thermophilus growth in Phase 4 and In-sure (i.e.
a commercially available BIM) was influenced by two factors: (a) the age of the
culture, for example, the starter cultures lost their activity in milk after 16–24 hour,
whilst in BIM retained their activity for 40–48 hours, and (b) the heat treatment
used for preparation of the BIM and agitation during growth affected S. thermophilus activity in relation to the BIM used (i.e. In-sure but not Phase 4).
6.3.10.11 Preservatives
In some countries, the addition of preservatives (e.g. K- or Na-sorbate, benzoic acid
or nisin) is permitted in fruit yoghurt, but not in natural yoghurt (for details refer
to Section 2.7.2 in Chapter 2). These compounds are mycostatic agents and, at the
same time, they can affect the activity of the starter cultures (see Table 2.12; Gupta
and Prasad, 1988; Kebary and Kamaly, 1991; Rajmohan and Prasad, 1994).
6.3.10.12 Miscellaneous compounds
The concentration (mg l-1) of fatty acids (1000), ethylenedichloride and methylsuphone (10–100 each) and acetonitrile, chloroform or ether (10 each) had an
inhibitory effect on S. thermophilus (see also Tamime and Deeth, 1980;
Antonopoulou et al., 1996).
6.4
Conclusion
It is evident that milk is an excellent growth medium for yoghurt starter cultures,
but the rate of growth in milk is influenced by a multitude of factors. Thus, using
milk free from these inhibitory agents, providing hygienic standards during the
preparation of starter culture and production of yoghurt and using the right combination of S. thermophilus and L. delbrueckii subsp. bulgaricus will lead to successful growth. Future development of these cultures in terms of resistance against
bacteriophage and/or inhibitory agents will help to minimise culture failure during
production.
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6.5
423
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